Volume 123, Issue 1, Pages 25-35 (October 2005) Phosphodiesterase 4D Deficiency in the Ryanodine-Receptor Complex Promotes Heart Failure and Arrhythmias  Stephan E. Lehnart, Xander H.T. Wehrens, Steven Reiken, Sunita Warrier, Andriy E. Belevych, Robert D. Harvey, Wito Richter, S.-L. Catherine Jin, Marco Conti, Andrew R. Marks  Cell  Volume 123, Issue 1, Pages 25-35 (October 2005) DOI: 10.1016/j.cell.2005.07.030 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 PDE4D Deficiency Promotes Age-Related Cardiomyopathy *p < 0.05 versus wt; #p < 0.05 versus PDE4D−/−. (A) Echocardiography at 3 month intervals showing progressive increase in left ventricular end-diastolic diameter (LVEDD) in PDE4D−/− mice (open squares, wt; filled squares, PDE4D−/−; n = 12 each). Data in (A)–(D) are mean ± SEM. (B) Age-dependent increase in heart-to-body-weight ratio (HW/BW) in PDE4D−/− mice (open bar, wt; filled bar, PDE4D−/−). (C) Age-dependent decrease in ejection fraction (EF) in PDE4D−/− mice (open bar, wt; filled bar, PDE4D−/−). (D) Reduced cardiac contractility (dP/dt/Pid) in PDE4D−/− mice at 3, 9, and 15 months of age (open squares, wt; filled squares, PDE4D−/−). (E) Histology showing dilated cardiomyopathy in PDE4D-deficient mouse hearts. Cell 2005 123, 25-35DOI: (10.1016/j.cell.2005.07.030) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Normal cAMP and β-Adrenergic-Receptor Levels in PDE4D−/− Mouse Hearts and Increased cAMP Levels at the Z Lines Detected by FRET-PKA (A) cAMP concentrations were not significantly increased in hearts of 3- to 6-month-old PDE4D−/− mice (wt, n = 5; PDE4D−/−, n = 5; p = NS). Each heart was extracted and assayed separately in quadruplicate experiments. Data in (A), (B), and (E) are mean ± SD. (B) β-adrenergic-receptor density was unchanged in PDE4D−/− mice (wt, n = 5; PDE4D−/−, n = 5; p = NS). (C) Comparison of the Bmax values for β-adrenergic-receptor density calculated separately for each of the five wt or five PDE4D−/− knockout mice investigated. (D) FRET-PKA showing increased cAMP-dependent signal over the Z lines (site of localization of RyR2) after low-dose isoproterenol stimulation in PDE4D−/− cardiomyocyte when compared to wild-type cardiomyocyte (white and black areas represent sensing of highest and lowest cAMP concentrations, respectively). (E) Bar graph summarizes Z line intensity-profile analysis of 480 nm/545 nm intensity ratio from five wt and six PDE4D−/− cells (*p < 0.05). Dimensions as indicated. Cell 2005 123, 25-35DOI: (10.1016/j.cell.2005.07.030) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Age-Dependent Alterations in RyR2-Channel Complex and Function in PDE4D−/− Murine Heart (A) Immunoblots showing progressive increase in PKA phosphorylation of RyR2 at Ser2808 (second panel) but no change in CaMKII phosphorylation at Ser2814 (third panel) and progressive decrease in calstabin2 binding to the RyR2 complex (fourth panel). PKA catalytic (-C) and regulatory (-RII) subunits are not changed during heart failure (HF) development (fifth and six panels). (B) Quantification of age-dependent increase in RyR2 PKA phosphorylation (open bars, wt; filled bars, PDE4D−/−; *p < 0.05 versus wt, #p < 0.05 versus PDE4D−/−). Data in (B)–(E) and (G) are mean ± SD. (C) Quantification of age-dependent decrease in calstabin2 in the RyR2 complex (open bars, wt; filled bars, PDE4D−/−; *p < 0.05 versus wt, #p < 0.05 versus PDE4D−/−). (D) Quantification of CaMKII-specific RyR2 phosphorylation showing no significant changes with aging. (E) Immunoprecipitation of RyR2 showing complete absence of PDE activity in the RyR2 complex in PDE4D−/− mouse hearts. *p < 0.05 versus wt; #p < 0.05 versus untreated wt. Rol, rolipram; Mil, milrinone. (F) Single-channel recordings of wt (left) and PDE4D−/− RyR2 (right) at 3 months of age (top) and 15 months of age (bottom). Current trace from 3-month-old PDE4D−/− heart shows slightly increased channel open probability (Po) with short openings consistent with increased PKA phosphorylation. At 15 months of age, RyR2 channels from PDE4D−/− hearts showed significantly increased Po and subconductance states (indicated by dotted lines), as evidenced by current-amplitude histograms. Upper traces represent 5 s; lower traces represent 500 ms. Channel openings are upward; full openings are 4 pA; closed state is indicated by “c.” (G) Bar graphs summarizing Po, open frequency (Fo), average open time (To), and average closed time (Tc) at 15 months of age in wt and PDE4D−/− groups. *p < 0.001; n = 10 each. Cell 2005 123, 25-35DOI: (10.1016/j.cell.2005.07.030) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 PDE4D3 Is a Component of the RyR2 Ca2+-Release-Channel Complex (A) PAN-PDE4 antibody against the conserved UCR2 domain (α-4PAN, top panel) and antibody raised against the N-terminal domain unique to PDE4D3 (α-4D3, bottom panel) were used to detect PDE4D isoforms in extracts of COS7 cells overexpressing recombinant PDE4D splice variants 1 to 9. Samples were size fractionated on 6% SDS-PAGE and blotted onto Immobilon-P membranes. (B) Immunoblotting with splice-variant-specific anti-PDE4D3, anti-PDE4D8, and anti-PDE4D9 antibodies shows expression of all three major forms in the heart. However, immunoprecipitation of RyR2 followed by splice-variant-specific immunoblot demonstrates that only PDE4D3 is associated with RyR2. Reverse immunoprecipitation with a specific anti-PDE4D3 antibody confirms that PDE4D3 is physically associated with RyR2. (C) Immunoblots of cardiac lysates showing amounts of RyR2 and PDE4D3 in wt, PDE4D+/−, and PDE4D−/− mice. Bar graph shows a 37% reduction of PDE4D3 in the RyR2 complex in cardiac lysates of PDE4D+/− mice relative to wt. *p < 0.05, n = 3 for each genotype. Data in (C) and (D) are mean ± SD. (D) Coimmunoprecipitation using anti-RyR2 antibody, showing a 44% decrease of PDE4D3 bound to RyR2 in PDE4D+/− mouse hearts relative to wt. *p < 0.05, n = 3 for each genotype. Cell 2005 123, 25-35DOI: (10.1016/j.cell.2005.07.030) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Reduced PDE4D3 in the RyR2 Complex in Human Heart Failure (A) PDE4D3 was detected in human cardiac lysate and in the immunoprecipitated RyR2 complex (IP:RyR2); IP:IgG, negative control. (B) RyR2 was immunoprecipitated from cardiac homogenates of normal human (N) and heart failure (HF) samples. PDE4D3 binding to RyR2 was significantly decreased in human HF. Increased PKA phosphorylation was detected by a phosphoepitope-specific RyR2-Ser2808 antibody in HF samples. (C) RyR2 bound PDE4D3 activity was significantly decreased in HF, as evidenced by close-proximity substrate cAMP catalysis (#p < 0.001). Data in (C) and (D) are mean ± SD. (D) Rolipram (R), a PDE4-specific inhibitor, but not milrinone (M), a PDE3-specific inhibitor, decreased RyR2-associated PDE activity submaximally; C, control (#p < 0.001 versus untreated sample). Cell 2005 123, 25-35DOI: (10.1016/j.cell.2005.07.030) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Cardiac Arrhythmias due to PDE4D3 Inhibition Are Suppressed in Mice Harboring Mutant RyR2 that Cannot be PKA Phosphorylated (A) Susceptibility to exercise-induced sustained ventricular arrhythmias (sVT) and nonsustained ventricular arrhythmias (nsVT) was significantly increased in PDE4D−/− compared to wt mice (each n = 6, *p < 0.05). (B) Rolipram (0.3 mg/kg body weight) maximally increased RyR2-Ser2808 PKA phosphorylation during exercise in vivo. Treatment as indicated on top: Rol, rolipram; Epi, epinephrine (0.1 mg/kg); white bars, no rolipram; black bar, rolipram-treated mice; *p < 0.05 between treatments in wt mice; #p < 0.001 wt versus homozygous RyR2-S2808A knockin (S2808A+/+) mice. Control mice were treated with placebo (rolipram carrier, 0.5% DMSO). Data are mean ± SD. (C) In rolipram-treated mice, susceptibility to exercise-induced sustained ventricular arrhythmias (sVT) and nonsustained ventricular arrhythmias (nsVT) was significantly decreased in homozygous RyR2-S2808A knockin (S2808A+/+) mice compared to wt mice (each n = 6, #p < 0.05). (D) Mortality from sudden death was significantly increased in PDE4D+/− mice 24 and 72 hr after MI compared to wt mice. White bars, wt mice; black bars, PDE4D+/− mice. *p < 0.01 between wt and PDE4D+/− groups. Cell 2005 123, 25-35DOI: (10.1016/j.cell.2005.07.030) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 PDE4D3 Deficiency Promotes HF Progression (A) LVEDD increased in PDE4D+/− (black squares) compared to wt (open squares) mice before (control, CO) and 14 and 28 days after myocardial infarction (MI) (*p < 0.05 versus wt). Both treatment with the 1,4-benzothiazepine JTV-519 (red line), which enhances calstabin2 binding to RyR2, or crossing the PDE4D+/− mice with RyR2-S2808A mice that harbor RyR2 that cannot be PKA phosphorylated (green line) significantly reduced the remodeling of the left ventricle following MI (#p < 0.01 versus PDE4D+/−). Data in (A)–(C) are mean ± SEM. (B) Reduced cardiac EF in PDE4D+/− mice 28 days after MI. wt, open bars; PDE4D+/− mice, filled bars (*p < 0.05 versus CO same genotype). Both treatment with the 1,4-benzothiazepine JTV-519 (red bar), which enhances calstabin2 binding to RyR2, or crossing the PDE4D+/− mice with RyR2-S2808A mice that harbor RyR2 that cannot be PKA phosphorylated (green bar) significantly improved left ventricular EF following MI (#p < 0.01 versus PDE4D+/− MI). (C) Left ventricular contractility (dP/dt)/Pid normalized to 100% of control in wt (open bars) and haploinsufficient PDE4D+/− mice (black bars) before (control, CO) and 28 days after MI (*p < 0.05 versus CO in same genotype). Treatment with JTV-519 (red bar) or crossing the PDE4D+/− mice with RyR2-S2808A mice (green bar) significantly improved contractility following MI (#p < 0.01 versus PDE4D+/− MI). (D) Immunoblot showing levels of PDE4D3, calstabin2, and RyR2-Ser2808 PKA phosphorylation in the immunoprecipitated RyR2-channel complex. JTV-519 treatment (red) or crossing the PDE4D+/− mice with RyR2-S2808A mice (green) significantly increased the binding of calstabin2 to RyR2 in the PDE4D-deficient mice. (E) Quantification of RyR2 PKA phosphorylation in wt and PDE4D+/− mice before (CO) and 28 days after MI, showing significantly more RyR2 PKA hyperphosphorylation in PDE4D+/− mice (*p < 0.05 versus wt at same time point). Treatment with JTV-519 (red bar) or crossing the PDE4D+/− mice with RyR2-S2808A mice (green bar) significantly reduced RyR2 PKA phosphorylation following MI (#p < 0.01 versus PDE4D+/− MI). Data in (E) and (F) are mean ± SD. (F) Quantification of RyR2-associated PDE activity showing significant decrease in PDE4D+/− mice 28 days before (CO) and after MI compared to wt (*p < 0.05 versus wt). Treatment with JTV-519 (red bar) or crossing the PDE4D+/− mice with RyR2-S2808A mice (green bar) significantly increased RyR2-associated PDE activity following MI (#p < 0.01 versus PDE4D+/− MI). Cell 2005 123, 25-35DOI: (10.1016/j.cell.2005.07.030) Copyright © 2005 Elsevier Inc. Terms and Conditions